Hazelbauef' Gerald L Program Director/Principal Investigator (Last, First, Middle): PROJECT SUMMARY (See instruc{ions): Receptors that mediate chemotaxis in Escherichia coli are paradigms for the large family of bacterial sensory receptors and a larger family of two-componenf' receptors. This application for an extension of my MERIT Award for its second five years focuses on functional mechanisms and structures of chemoreceptors and the signaling complexes they form. This focus is consistent with the NIGMS mission of basic research that increases understanding of life processes and lays the foundation for medical advances. Studies will emphasize E. coli chemoreceptors and utilize biochemical, mutational, biophysical, and structural approaches. Some approaches involve collaborative efforts with other research groups and most exploit advantages of chemoreceptors inserted in Nanodiscs, water-soluble plugs of lipid bilayer surrounded by a protein annulus, which are effective for manipulating bilayer-inserted receptors. Specific aims address issues central to understanding chemoreceptors. 1) Structure and conformational changes of signaling of the chemoreceptor dimer, the fundamental receptor organizational and functional unit. Receptor structure and conformational changes will be characterized using site-directed spin labeling and electron paramagnetic resonance spectroscopy, ONDP relaxometry, structuralelectron microscopy including high-resolution cryo-EM and X-ray diffraction. Results will define the structure of this crucial unit and identify fundamental mechanisms of receptor intra-molecular and transmembrane signaling. 2) Functional and structural properties of core signaling complexes, the fundamental unit of kinase activation and control. Functional characterization of soluble core signaling complexes will provide basic insights into its functional capabilities, allosteric interactions and kinase activity. Structural properties will be characterized by biophysical assays, advanced techniques of electron microscopy and, if preliminary results merit, X-ray crystallography. In summary, the proposed research aims to substantially advance our understanding of bacterial chemoreceptors, a paradigm for biological sensory signaling. (See instructions): ability to sense and respond to the environment is an essential biological process shared by all cells. investigate this process in bacteria, which are easier to manipulate than cells from higher organisms, the aim of discovering basic principles. Knowledge of these principles will aid understanding and of the many human health problems caused by defects in the ability to sense and respond.